My research activity focus on understanding the mechanisms that render macrophages resistant to infection by HIV, the virus responsible of AIDS pandemic. In particular, we focus on the activity of enzyme that is called some HT-1 that degrades the NTPs, the building blocks of the viral genomes. But this enzyme has also other activities that we try to elucidate.
We established a protocol to genetically engineer THP1 cells. These cells are a well established surrogate for human macrophages to study. For instance, the replication of HIV-1.
These cells are difficult to transfect to manipulate their protein content. Therefore, we establish methods to manipulate their genome using the CRISPR-Cas9 technology. From the design of SG RNAs up to the validation of polyclonal and monoclonal populations.
Our protocol aims to achieve high efficiency editing of a gene of interest in THP1 cells that will be electroporated with preassembled ribonucleoprotein complexes made by the Cas9 nuclease energy RNAs. In this approach, Cas9 activity is limited in time. This reduces the possibility to generate of target effects that often arise when lenti vectors are employed.
And in this case, indeed Cas9 expression is prolonging time and therefore it can be damaging for the cell. To begin fill each well of a 24 well culture plate with 500 microliters of RPMI 1640 medium without antibiotic and supplemented with 20%heat inactivated and filtered FBS. Place the plate in a humidified incubator overnight.
To rehydrate the dry single guide or sgRNAs to a final concentration of 100 micromolar add 10 microliters of TE buffer per one nano mol of sgRNA. Vortex the solution for 30 seconds and incubate at four degrees Celsius overnight to ensure complete rehydration. The next day, to prepare the 30 micromolar working sgRNA solution, first briefly homogenize the 100 micromolar stock sgRNA solution with a pipette, then dilute the stalk in nuclease free water.
Vortex the solution for 30 seconds and incubate at room temperature for five minutes. Dilute one microliter of each of the three, 30 micromolar sgRNA's working solutions and 0.5 microliters of 20 micromolar Cas9 solution in 3.5 microliters of resuspension buffer R, to assemble the Cas9 sgRNA ribonucleoproteins add a one to nine molar ratio. Vortex briefly and incubate for five minutes at room temperature.
To prepare an unedited control add 0.5 microliters of 20 micromolar Cas9 solution to 6.5 microliters of resuspension buffer R.Vortex briefly and incubate for five minutes at room temperature, add five microliters of resuspension buffer R to samples to achieve a final volume of 12 microliters per electroporation condition. To prepare THP-1 cells after cell counting, collect a volume equivalent to 0.2 times 10 to the power of six of THP-1 cells and centrifuge them at 336G for five minutes at 20 degrees Celsius, aspirate the supernatant using a pipette and resuspend the pellet in 500 microliters of PBS. Centrifuge again and resuspend the cell pellet in the 12 microliters of ribonucleoprotein solution.
To set up the electroporation system, place the pipette station under a biosafety cabinet and position an electroporation tube inside the cabinet, add three milliliters of buffer E from the electroporation kit to the electroporation tube. Turn on the electroporation device and use the touchscreen to set the electroporation parameters, voltage to 1, 500 volts. Duration to 10 milliseconds.
And number to three. Equip the electroporation pipette with a tip and aspirate 10 microliters of the ribonucleoprotein THP-1 solution. Insert the pipette into the electroporation tube and press start on the electroporation device screen.
Wait for the message complete to appear and remove the pipette from the tube. Transfer the THP-1 cells into a well of the preheated 24 well culture plate and gently homogenize the sample. Place the plate back into the humidified incubator and allow the cells to rest undisturbed for 72 hours.
After electroporation and cell counting, withdraw a volume equivalent to 0.1 times 10 to the power of six THP-1 cells into a 1.5 milliliter tube. Centrifuge the cells at 336G for five minutes at 20 degrees Celsius and aspirate the supernatant before resuspending the pellet in 500 microliters of PBS. Again, centrifuge the cells, aspirate as much supernatant as possible without disturbing the pellet and snap, freeze the pellet.
For the extraction of genomic DNA resuspend the pellet with 50 microliters of DNA extraction solution. Homogenize it and transfer the entire volume to a 0.2 milliliter PCR tube. Vortex the tube and centrifuge it briefly for pulse of three seconds.
Place the tube in a thermal cycler and heat at 65 degrees Celsius for 15 minutes, followed by 98 degrees Celsius for 10 minutes. Next, dilute the extracted DNA with 90 microliters of Ultrapure water, vortex and centrifuge briefly at 5000G for three seconds. Dilute the PCR primer in ultrapure water to a final concentration of 10 micromoles.
Prepare the PCR mix in a 0.2 milliliter PCR tube. Place the PCR tubes in the thermal cycler and run the program with the specified settings. For polyclonal edited populations in a new 0.2 milliliter tube.
Mix 17.5 microliters of the PCR amplicon with two microliters of E buffer two for a final volume of 19.5 microliters. Vortex the solution and centrifuge briefly, alternatively, for screening single cell clones mix the PCR products from the edited and unedited control cells in a one-to-one ratio. For hetero duplex formation place the tubes in a thermal cycler and run the given program.
After the hetero duplex formation add 0.5 microliters of T seven endonuclease one solution to the tube and incubate at 37 degrees Celsius for 30 minutes. To prepare the samples for gel electrophoresis, mix five microliters of the T7 endonuclease one digestion products or undigested PCR product with five microliters of water and two microliters of six xDNA loading dye, load the samples and size ladder into the gel and run the gel at 80 volts for 45 minutes. CRISPR Cas9 mediated gene editing of the EGFP locus resulted in a sharp decline of GFP positive cells reaching more than 90%reduction by day seven post electroporation.
By day three post electroporation, cell numbers had halved due to electroporation induced stress, but fully recovered by day seven in complete RPMI medium. T7 Endonuclease one say and Agarose's gel electrophoresis demonstrated the loss of approximately 75 base pair fragments in edited cells confirming CRISPR editing of the EGFP target site.
